Successful dissemination of HIV-1 in infected individuals depends on efficient transmission of viral particles from infected (producer) to uninfected (target) cells. In vitro propagation studies have established that HIV-1 particles are most effectively transmitted to target cells if they bud into the cleft of the so-called virological synapse (VS) that forms between producer and target cells. The events leading to the formation, maintenance and disassembly of this synapse are poorly understood. Previously, we and other laboratories have established that HIV-1 particles exit from cells at sites that are enriched in tetraspanins. These cellular membrane proteins normally function as organizers of plasma membrane-based processes, including cell-cell fusion and adhesion. Our preliminary data suggest that individual members of the tetraspanin family are not required for virus release, but they point to a role of these proteins in regulating virus transfer to target cells. We thus propose to evaluate if tetraspanins in producer cells are regulatory cofactors necessary for efficient cell-to-cell transmission of HIV-1 particles. We will also analyze the effects of virus-mediated downregulation of tetraspanins. Altogether, these studies will provide further insight into the molecular mechanisms underlying HIV-1 spread and thus pathogenesis. In the Specific Aims of this proposal we propose: 1) To test the hypothesis that tetraspanins inhibit HIV-1-induced membrane fusion, thus allowing cell-to-cell transfer without fusion of producer and target cell. 2) To determine if tetraspanins in HIV-1 producer cells promote efficient transmission of viral particles to target cells by supporting the formation, the organization and the disassembly of the VS. 3) To examine the kinetics, determinants and potential consequences of HIV-1-induced tetraspanin downregulation in infected cells.